PROJECT SUMMARY The coronavirus disease 2019 (Covid-19) pandemic erupted into the midst of the drug overdose crisis in the US. Over 60% of drug overdoses are linked to opioid analgesics, with repeated overdoses indicative of the development of opioid use disorder (OUD), a growing and life-threatening medical condition. Given that we are unable to decouple opioid-induced pain relief from opioid abuse and OUD at present, opioid analgesics will remain essential in medicine and their use is likely to co-occur with Covid-19 illness. Opioid medications generally enhance viral replication, pathogenesis, and infectious disease progression, in part via weakening the immune system. The SARS-CoV-1 virus is neuroinvasive and neurovirulent and is linked to progressive central nervous system (CNS) symptoms (e.g., confusion, delirium, dysphoria), aligning with symptomatology seen in some Covid-19 patients. As for other viruses, SARS-CoV-2 immunopathology and disease progression will prove to be a function of virus pathogenesis and host vulnerability, one of which may be chronic opioid use in pain management and/or OUD. These findings inspire the urgent need to understand how opioids impact specific host vulnerability factors and cofactors, and how medications proposed to treat Covid-19 affect opioid signaling, particularly within single cells that control CNS actions of opioids. Initial observations suggest that host factors involved in SARS-CoV-2 cellular entry are altered in the brains of heroin users. We recently reported that brain- region speci?c expression of immune cytokines and chemokines tracks with lifetime fentanyl intake in rats, suggesting that opioid exposure regulates host viral entry and immune function relevant to Covid-19 infection. Given the concurrent Covid-19 and overdose crises, the chronic use of opioids in pain management, the growing problem of OUD as well as illegal abuse of opioid drugs, we propose two specific aims of immediate relevance to predicting Covid-19 disease severity and appropriate therapeutics in opioid-exposed OUD patients. Based upon this premise, we will test the hypothesis that withdrawal from chronic fentanyl self-administration in male and female rats will impact key targets that mediate SARS-CoV-2 infection in identified single cells in CNS via single-nuclei RNAseq transcriptomics (Aim 1) and that acute or chronic Covid-19 medication candidates will alter signaling profiles of opioids (e.g., fentanyl, buprenorphine) in a cellular system (Aim 2). We expect to discover altered markers of host viral entry and immunological status in identified single CNS cells following chronic fentanyl self-administration, establishing the potential for enhanced neurological damage in Covid-19 patients. We also expect to demonstrate interactions between Covid-19 medications and opioid signaling which predict potential interactions in vivo. These innovative aims are consistent with the goals of NOT-DA-20-047 to determine if opioid exposure is a risk factor for the onset and progression of Covid-19.